C H A P T E R
32
-Oryzanol: An Attractive Bioactive
Component from Rice Bran
Christelle Lemus, Apostolis Angelis, Maria Halabalaki, Alexios Leandros
Skaltsounis
University of Athens, Division of Pharmacognosy and Natural Products Chemistry, Department of Pharmacy,
Athens, Greece
INTRODUCTION
Oryza sativa L. seeds of the Poaceae family, commonly
known as Asian rice or simply rice, could be consid-
ered as one of the most important grains since it is con-
sumed by the half of the world population. According
to a United States Department of Agriculture (USDA)
report,1 453.4 million tonnes of rice was consumed
worldwide in 2010/2011.
In Asia in particular rice comprises a basic compo-
nent of the daily diet,2 and it occupies one of the top
positions of commodities with the highest worldwide
production according to FAOSTAT (the Statistics Divi-
sion of the Food and Agriculture Organization of the
United Nations).2 As expected, Asia is by far the main
producer and China the most representative country,
producing 200 million tonnes of rice and paddy in 2010
(Fig. 32.1).
The oil deriving from rice, namely rice bran oil
(RBO), is one of the most commonly used cooking oils
in Asia, and the major by-product of the rice milling
industry. RBO represents a rich source of high added-
value phytochemicals of nutritional, pharmaceutical,
and cosmetic interest, such as tocopherols and tocot-
rienols (tocols or vitamin E), lecithin, carotenoids, and
-oryzanol, and has therefore attracted considerable
interest in recent years.3,4 -Oryzanol is of particular
significance because it is abundant in RBO compared to
other vegetable oils.5
-Oryzanol was first isolated in 1954 by Kaneko and
Tsuchiya6 from the unsaponifiable fraction of rice bran as
a crystalline substance; it was considered to be a single
compound and was named oryzanol in reference to the
botanical species of rice, Oryza sativa. Three years later,
Wheat and Rice in Disease Prevention and Health
http://dx.doi.org/10.1016/B978-0-12-401716-0.00032-5 2014 Elsevier Inc. All rights reserved.
Shimizu etal.,7 using a different extraction method, dis-
covered that -oryzanol was not a single entity and sug-
gested that it was composed of three ferulic acid esters
of triterpene alcohols, which were designated oryza-
nol A, B, and C. Subsequently the same scientific team
identified oryzanol A as cycloartenyl ferulate,8 and in
1958 oryzanol C was identified as 24-methylene cyclo-
artanyl ferulate.9 Finally, oryzanol B was found to be a
mixture of oryzanol A and C. Since then, various studies
have been carried out investigating the composition of
-oryzanol, allowing the identification of more than 20
ferulic acid esters of triterpene alcohols and sterols by
different methods.10
Given that RBO is an important by-product in the
rice industry, much attention has been paid to opti-
mization of its production and further treatment pro-
cesses. In the standard industrial procedure of RBO
production a refining process takes place through a
deacidification treatment with alkali, giving rise to
soapstock. This residue is quite valuable, since the
highest percentage of -oryzanol contained in the
crude oil is lost in the soapstock (83%95%, according
to Krishna etal.11). Therefore soapstoack is the richest
source of -oryzanol and probably the most important
by-product of rice processing.
-Oryzanol generally exhibits a wide spectrum of
health-beneficial effects, including anticarginogenic,
anti-inflammatory, antihyperlipidemic, antidiabetic, and
neuroprotective, which are mainly attributed to its anti-
oxidant capacity.4 This pleiotropic biological profile of
-oryzanol, together with its high availability in indus-
trial by-products, has contributed significantly to the
observed increasing interest from academia and indus-
try, especially in recent years.
409
410 32. -ORYZANOL: ANALYSIS AND CHARACTERIZATION

FIGURE 32.1 (A) Whole world production: proportion of rice amongst other grains (average 19612010). (B) Production of top five produc-
ers of rice (average 19612010). Food and Agriculture Organization of the United Nations (http://faostat3.fao.org/home/index.html#VISUALIZE).
Please see color plate at the back of the book.

21 22 21 22
23 25 26 23 25 26
18 24 18 24
20 20
12 12
11 17 11 17
19 13 27 19 13 27
1 16 1 16
9 14 9 14
2 2
O 10 8 15 O 10 8 15

4 7 4 7
O 3 5 O 3 5
6 6
RO 29 28 RO

HO Esters of triterpene alcohols HO Esters of phytosterols

R = Me : Steryl ferulates
R = H : Steryl caffeates

FIGURE 32.2 General structures and numeration of -oryzanol compounds.

CLASSIFICATION AND BIOSYNTHESIS With regard to the structural composition, it seems

OF -ORYZANOL clear that steryl ferulates biogenetically originated
from the esterification of ferulic acid with sterols and/
Chemically, -oryzanol is a mixture of structurally or triterpene alcohols. The steryl ferulates principally
related components, and specifically esters of ferulic acid differ at the triterpenoid moiety, which represents the
with phytosterols and triterpene alcohols, also referred bulkier part of the molecule, while both sterols and
as steryl ferulates. Steryl ferulates have been identi- triterpene alcohols consist of a four-ring cyclopentano-
fied in rye, corn, triticale, barley, and wheat, but their phenanthrene skeleton with a hydroxyl group at posi-
richest source is rice, and specifically RBO.12 The most tion 3. Their biosynthesis initiates with acetyl-CoA and
abundant steryl ferulates in RBO, comprising almost implicates the enzymatic controlled formation of iso-
95% of -oryzanol, are campesterol, -sitosterol, cyclo- pentenyl diphosphate (IPP) via mevalonic acid. IPP is
artenol, and 24-methylenecycloartenol esters (Fig. 32.2). then involved in the production of prenyl diphosphate
Although the constituents of -oryzanol were identified homologues (DMAPP, GPP) by a succession of elonga-
as steryl esters of ferulic acid, Fangs work has recently tion reactions. Condensation of three molecules of IPP
revealed the presence of caffeate esters of cycloartenol from geranyl diphosphate (GPP) produces squalene,
and campesterol as well.13 which is epoxided to oxidosqualene, a common precur-
Numerous others steryl ferulates have been reported sor of sterols and triterpene alcohol14 (Fig. 32.3). Several
to be present in -oryzanol in low concentrations, labeled experiments led to the determination of path-
detected and identified via several spectroscopic meth- ways involved in triterpene biogenesis via lanosterol or
ods, and most of these are presented in Table 32.1. cycloartenol.15

A. OVERVIEW OF RICE AND HEALTH

 CLASSIFICATION AND BIOSYNTHESIS OF -ORYZANOL 411
TABLE 32.1Basic -Oryzanol Components Identified Using MS and NMR Techniques
R1 R2 Name Formula M (g/mol) Ref(s)

R2 F 24-Methylenecycloartanyl C41H60O4 616.45 Fang etal.13, Akihisa etal.22,

ferulate Xu and Godber75, Berger
H etal.187

F Cycloartenyl ferulate C40H58O4 602.43 Fang etal.13, Akihisa etal.22,

R1O
H Xu and Godber75, Berger
etal.187

C Cycloartenyl caffeate C39H56O4 588.42 Fang etal.13

F Cycloartanyl ferulate C40H60O4 604.45 Fang etal.13

F Cyclobranyl ferulate C41H60O4 616.45 Berger etal.187

F OH (24)-cycloart-25-ene-3, C39H56O5 604.41 Angelis etal.17

24-diol-3-trans ferulate

F OH Cycloart-23-Z-ene-3, C39H56O5 604.41 Angelis etal.17

25-diol-3-trans ferulate

R2 F Cycloeucalenyl ferulate C40H58O4 602.43 Fang etal.13, Akihisa etal.22

R1O
H

R2 F Campesteryl ferulate C38H56O4 576.42 Fang etal.13, Xu and Godber75,

Berger etal.187
H
C Campesteryl caffeate C37H54O4 562.40 Fang etal.13
H
R1O F Sitosteryl ferulate C39H58O4 590.43 Fang etal.13, Akihisa etal.22,
Xu and Godber75, Berger
etal.187

F Stigmasteryl ferulate C39H56O4 588.42 Fang etal.13, Akihisa etal.22,

Xu and Godber75, Berger
etal.187

R2 F 7-Stigmastenyl ferulate C39H56O4 588.42 Xu and Godber75

H
R1O
F 7-Sitostenyl ferulate C39H58O4 590.43 Fang etal.13, Xu and Godber75
H

F 7-Campestenyl ferulate C39H56O4 576.42 Fang etal.13, Xu and Godber75

R2 F Gramisteryl ferulate C39H56O4 588.42 Akihisa etal.22

H F Citrostadienyl ferulate C40H58O4 602.43 Akihisa etal.22

R1O
H

(Continued)

A. OVERVIEW OF RICE AND HEALTH

412 32. -ORYZANOL: ANALYSIS AND CHARACTERIZATION

TABLE 32.1Basic -Oryzanol Components Identified Using MS and NMR Techniquescontd

R1 R2 Name Formula M (g/mol) Ref(s)

R2 F Campestanyl ferulate C38H58O4 578.43 Fang etal.13, Xu and Godber75

H F Sitostanyl ferulate C39H60O4 592.45 Fang etal.13, Akihisa etal.22,

R1O
H Xu and Godber75

O O
MeO HO
F= C=
HO HO

Ferulate Caffeate

O O HO O HO
CoA
S HO OH HO OP
Acetyl-CoA Mevalonic acid (MVA) Mevalonic acid 5-phosphate (MVAP)

O HO
IPP isomerase
OP P OP P HO OP P

Dimethylallyl diphosphate (DMAPP) Isopentenyl diphosphate (IPP) Mevalonic acid 5-diphosphate (MVAPP)

OP P

Geranyl diphosphate (GPP)

HO
Lanosterol
IPP x 3

Triterpene alcohols
OP P Sterols

Squalene O Oxidosqualene

O O
P = P O P O
O O HO
Cycloartenol

FIGURE 32.3 Biogenetic synthesis of sterols.

Structurally, the number of methyl groups attached
on C-4 allows differentiation of the sterol ferulates from
the triterpene alcohol ferulates, the former having no
methyl or just one methyl (4-methylsterol) group on
C-4, and the latter containing two methyl groups at the
tetracyclic ring system. In the case of triterpene alcohol
ferulate esters, a cyclopropane in position C-9/C-10 is
also usually present.16 The composition of the side chain
of sterols and triterpene alcohols of -oryzanol varies
with the different derivatives (Table 32.1). Recently, a
new group of -oryzanol, designated as polar -oryzanol
has been suggested by Angelis etal. and generally it con-
cerns hydroxylated derivatives characterized by alkyl
groups attached to C-24. Specifically, three hydroxylated
triterpene alcohol ferulates ([24R] and [24S]-cycloart-
25-ene-3,24-diol-3-trans ferulate, and cycloart-23Z-
ene-3,25-diol-3-trans ferulate) have been isolated and
identified.17 The presence of such hydroxylated ferulates
was also mentioned previously by Fang etal.13
The -oryzanol ferulates are characterized by
the trans configuration of the ferulic acid part.
Ferulic acid (FA) is a derivative of cinnamic acid

A. OVERVIEW OF RICE AND HEALTH

 EXTRACTION AND ISOLATION OF -ORYZANOL
COOH
NH2
OH
Tyrosine
COOH COOH
NH2 p-Coumaricacid
Phenylalanine Cinnamic acid
FIGURE 32.4 Biogenetic formation of ferulic acid.
(4-hydroxy-3-methoxycinnamic acid), and is one of the
most abundant phenolic acids in plants. FA is rarely
found in its free form, but is generally linked to sug-
ars, polysaccharides, or proteins.18 This moiety is also
responsible for the characteristic ultraviolet absorp-
tion observed in RBO. Biogenetically, this phenolic unit
comes from the methoxylation of the m-hydroxyl group
of caffeic acid, catalyzed by the enzyme O-methyltrans-
ferase.19 This suggests that the enzyme involved in the
biosynthesis of steryl ferulates could accept caffeic acid
as a substrate and thus explain the presence of caffeate
esters in -oryzanol (Fig. 32.4). As with many natural
phenols, FA possesses antioxidant activity,20 and has
been found to exhibit beneficial effects against cancer,
diabetes, and Alzheimer disease.21 Although the steryl
ferulates are generally characterized by a trans configu-
ration of the ferulic acid, several authors have also shown
the presence of cis derivatives.13,17,22 However, Fang etal.
suggest the possibility that these cis derivatives might
be artifacts due to a cistrans isomerization which may
occur during the production of rice bran because of the
long wavelength UV radiation.13
As mentioned previously, -oryzanol compounds
are formed through esterification between ferulic (or
caffeic) acid and a triterpene (sterol or triterpene alco-
hol). In order to investigate this hypothesis, Sato23
performed synthesis of the -oryzanol steryl ferulates
(14C labeled), starting with the formulation of guaia-
col with 14C-labeled formaldehyde to give vanillin-14C.
Condensation of this latter with malonic acid followed
by acetylation gave 4-acetylferulic acid-14C, which
was treated with 2SOCl2 forming 4-acetylferulic acid
chloride-14C. The nucleophilic reaction between the triter-
penyl alcohols (obtained by saponification of -oryzanol)
and the chloride-14C produced -oryzanol-14C with
A. OVERVIEW OF RICE AND HEALTH
413
COOH COOH COOH
O-Methyltransferase
OH OMe
OH OH OH
Caffeic acid Ferulic acid
a yield of 97% after crystallization. Akihisa22 has also
proposed a synthesis of eight steryl ferulates contained
in -oryzanol. Thus, 4-propionyl ferulate, obtained by
condensation of trans-ferulic acid and propionic anhy-
dride in an alkaline medium, was added to stigmasterol,
yielding a 4-propionyl ferulate of stigmasterol. Finally,
basic hydrolysis of the propionyl ester moiety gave
trans-stigmasteryl ferulate. Using the same synthetic
pathway, trans-gramisteryl ferulate and trans-citrostadi-
enyl ferulate were prepared from the corresponding free
sterols. It should be mentioned that the author has also
prepared the corresponding cis derivatives, by irradia-
tion at 365nm, under N2 with a 100-W mercury vapor
discharge lamp, of the trans-ferulates. In this manner,
cis-stigmasteryl ferulate, cis-gramisteryl ferulate, cis-
citrostadienyl ferulate, cis-cycloeucalenyl ferulate, and
cis-24-methylenecycloartanyl ferulate were synthesized.
It is important to note that recently a patent has been filed
concerning the preparation of steryl ferulates.24 The syn-
thesis involves the acetylation of ferulic acid followed by
esterification with a phytosterol in the presence of N,N-
dicyclohexylcarbodiimide (DCC) and 4-dimethylami-
nopyridine (DMAP). Finally, deprotection of the acetate
gave the desired phytosteryl ferulate.
EXTRACTION AND ISOLATION
OF -ORYZANOL
Rice bran comprises the outer layers of the brown rice
kernel (pericarp, tegmen, aleurone, and sub-aleurone)
and is obtained as a by-product of the rice milling pro-
cess. Rice bran constitutes approximately 810% of paddy
rice, which leads in huge production of this added-value
material globally.25 The high nutritious value of rice bran
414 32. -ORYZANOL: ANALYSIS AND CHARACTERIZATION
is due to its high contents of protein (1416%), dietary
fiber (812%), phytic acid (8.7%), and oil (1623%). The
extraction of rice bran leads to RBO, which contains an
important amount of biologically active nutraceuticals
such as -oryzanol, vitamin E (tocopherols and tocotri-
enols), and phytosterols.26,27 Apart from neutral lipids
(8890%), crude RBO contains a rich unsaponifiable frac-
tion (45%), free fatty acids (24%), and small amounts
of gum and waxes. The unsaponifiable fraction contains
mainly -oryzanol and tocols, while other bioactive
compounds, such as carotenoids, squalene, lecithin, tri-
cin, and long-chain alcohols, are also present in lower
concentrations.4,2830
The use of RBO as food product depends on the qual-
ity of the bran, the extraction method used, and the refin-
ing process. Rice bran contains several types of lipases
which are activated during the milling process, leading
to hydrolysis of lipids into free fatty acids (FFA). This
deterioration gives the products a rancid smell and bit-
ter taste, making them unsuitable for consumption.31
Although direct extraction of freshly milled rice bran
leads to good quality RBO, the commonly used proce-
dure includes initial stabilization of the bran prior to oil
extraction. The stabilization of rice bran aims to avoid the
process of hydrolytic rancidity by destruction or inhibi-
tion of lipases. A variety of methods, such as sun-drying,
cold storage, steaming, and ohmic heating, as well as
chemical treatments, has been used for this purpose. It
should be noted that the chosen method has a signifi-
cant effect on the lipases activity, the extraction yield,
and the levels of bioactive compounds.32,33 The stored
stabilized bran retains standard FFA levels for about
2 months, while food-grade n-hexane is the solvent of
choice for commercial extraction of RBO.3436 The major
advantage of this solvent is its high stability and capac-
ity for dissolving oil, and it is considered by FDA as safe
for extraction of edible oils from oil seeds or oil-bearing
materials.37 n-Hexane is mixed directly with the stabi-
lized rice bran in maceration or percolation type extrac-
tors, and the miscella, after removal of the solvent by
vacuum evaporation or distillation, yields crude RBO.
Although n-hexane is an effective solvent for extraction
of oil from rice bran, it poses some disadvantages, such
as toxicity at high concentrations and flammability, and
is also environmentally hazardous. For these reasons,
the bran oil industry has shown an increased interest in
alternative solvents as well as in solvent-free processes
such as ohmic heating and supercritical fluid extraction.
The crude RBO obtained from solvent extraction of
rice bran is unsuitable for edible use due to its high lev-
els of FFA, waxes, gums, and pigments. The removal of
these components by chemical or physical refining leads
to food-grade RBO. Chemical refining produces good
quality RBO regarding color and cloud point, and thus
is generally preferred over physical refining. However,
A. OVERVIEW OF RICE AND HEALTH
chemical refining causes significant losses of bioactive
components, which are removed into the soapstock,
in contrast to physical refining, which leads to RBO
rich in phytochemicals. Regarding -oryzanol content,
physical refining removes only 1.15.9% of -oryzanol
while chemical refining almost completely removes the
-oryzanol content (9395%) from crude RBO.11,38 The
beneficial properties of -oryzanol and tocopherols in
human health means that high commercial value has
been added to rice products. Thus, the factors that influ-
ence these phytochemicals extraction rates are of great
importance and have been extensively studied. More
than 50 research articles have been reported worldwide
investigating the main factors and their impact on the
levels of constituents of rice. Both genetic and environ-
mental factors seem to affect significantly the -oryzanol
content in rice and rice bran. Particular attention should
be given to the milling, extraction, and refining pro-
cesses due to the direct impact of those procedures on
-oryzanol levels.4,10 Generally, the -oryzanol content
in rice ranges significantly in different genotypes and
growing environments. Bergman and Xu reported a
study on seven rice cultivars of the Southern US over
2 years, and demonstrated that -oryzanols content
ranged from 2510 to 6864mg/kg, while the grow-
ing environment had a greater effect than the geno-
type.3 Another study, performed by Miller etal., on 30
brown rice cultivars of European origin grown at dif-
ferent areas and in different seasons showed approxi-
mately the same -oryzanol content (2663mg/100g)
and indicated that environmental conditions were the
main factor for this range. Moreover, it was found that
the degree of maturity of rice grains has no significant
influence on -oryzanol levels.39
On the other hand, Heinemann etal. analyzed 32
genotypes of brown rice belonging to the indica and
japonica subspecies, cultivated in Brazil, and found that
the mean content of -oryzanol across all samples was
significantly higher in japonica (246.3mg/kg) compared
to indica (190.1mg/kg) rice subspecies.40 Japonica vari-
eties were found to be richer in -oryzanol compared to
indica varieties, as demonstrated in a few more studies
conducted in commercial rice varieties growing in Tai-
wan and Pakistan. Beyond the above result the authors
found that: (1) no obvious difference in -oryzanol con-
tent was noted between black- and red-colored rice
varieties; (2) -oryzanol was affected significant by crop
year; and (3) basmati rice cultivars are a good source of
-oryzanol.4143
The impact of growing temperature on phytochemi-
cal levels was the subject of research reported by Britz
and coworkers. In this study, the seeds from six differ-
ent rice lines grown in replicate greenhouses were ana-
lyzed. The results showed that the -oryzanol levels
increased by 3557% at an elevated temperature in five
 EXTRACTION AND ISOLATION OF -ORYZANOL
of six lines, but the levels of tocols were not affected.
Moreover, the study demonstrated that the increased
temperature was the main factor altering the -oryzanol
content.44 On the contrary, the application of organic
and inorganic fertilizers and pesticides to the rice crop
had no particular impact on the content of -oryzanol
and vitamin E.45
Another critical factor that significantly modifies
the levels of several rice bioactive components is ger-
mination. The investigation of its influence and stud-
ies related to the development of functional foods have
been the subject of several published research articles.
For instance, Lee etal. found that during germination of
rough rice of four Korean varieties the -oryzanol con-
tent increased 0.8- to 1.5-fold,46 while Kim etal. reported
that the -oryzanol contents in rough rice and brown rice
were increased 1.13- and 1.20-fold, respectively, after
germination.47 The effect of germination time on the lev-
els of biochemical compositions and antioxidant activity
has been also explored. For instance, Moongngarm and
Khomphiphatkul showed that 2- to 3-day germination
of rough rice was the optimum time for the increase in
-oryzanol content and determined antioxidant activ-
ity,48 while Kiing etal. demonstrated that some Sarawak
cultivars are characterized by the highest concentration
of -oryzanol after 16 hours of germination.49
The change in -oryzanol, relative to other nutritional
components of paddy rice and brown rice during the
germination process, was also investigated by Oh and
coworkers. The results showed significant concentration
differences throughout germination, whilst brown rice
exhibited a higher -oryzanol content than paddy rice.50
Moreover, Sungsopha etal. investigated the influence of
germination and enzymatic treatment on the levels of the
bioactive contents and antioxidant activity of rice bran.
Among other findings, they discovered that both germi-
nation of rough rice and enzymatic treatment improved
the levels of -oryzanol two-fold, compared to untreated
rice bran.51
The milling process itself also seems to have a signifi-
cant impact on the levels of rice bioactive compounds.
Since the outer layers of the rice kernel contain almost
the total amount of -oryzanol in rice, the degree of
milling leads to products with different levels of these
compounds. Thus, the influence of the milling process
on the levels of -oryzanol has been reported in a few
studies. Overall, findings demonstrate that the con-
centrations of -oryzanol in rice, as well as the levels
of other compounds, are significantly decreased as the
degree of milling is increased.30,52 For instance, Tuncel
and Yilmaz showed that the -oryzanol content obtained
from different steps of milling procedure ranged from
12.19 to 3296.5mg/kg, while the whitening and polish-
ing processes led to the loss of approximately 94% of the
-oryzanol compared to brown rice.53
A. OVERVIEW OF RICE AND HEALTH
415
Critical factors for -oryzanol content, and rice bio-
actives in general, are the extraction and refining meth-
ods. These affect both the yield of the produced oil and
the levels of the biological active compounds signifi-
cantly. The most commonly used method for extraction
of RBO is solvent extraction, while soxhlet and super-
critical fluid extraction (SFE) are, so far, used mainly
in laboratory-scale production. Although n-hexane is
the solvent of choice, especially in large-scale extrac-
tion of RBO, numerous attempts have been made to find
alternative solvents that are able to increase the overall
yield of RBO and its phytochemical content. As for other
factors, the influence of the solvent and the extraction
method on the levels of -oryzanol has been extensively
investigated. For example, Xu and Godber compared
the ability of supercritical CO2 and organic solvents
to extract lipids and -oryzanol from rice bran. They
assayed several organic solvents and found that the mix-
ture of 50% n-hexane and 50% isopropanol (vol/vol), at
a temperature of 60C for 4560 minutes, produced the
highest yield (1.68mg/g of rice bran) of -oryzanol. On
the other hand, the use of SFE under a temperature of
50C and pressure of 68,901kPa (680atm) for 25 minutes
led to 5.39mg -oryzanol/g of rice bran an amount
approximately four times greater than the highest yield
achieved using solvent extraction. Moreover, it was pos-
sible to obtain an extract with a high concentration of
-oryzanol (5080%) after 1520 minutes of extraction
under optimized conditions.54
Along these lines, Imsanguan and coworkers
reported a comparative study in which the effects of
operating mode, temperature, pressure, and solvent on
-tocopherol and -oryzanol extraction yield from rice
bran were investigated. They also compared the effi-
ciency of SFE-CO2, solvent, and soxhlet extraction in
respect to the recovery of these compounds. The authors
found that the best conditions for SFE-CO2 extraction of
-oryzanol were 65C and 48MPa, in continuous operat-
ing mode, while, regarding solvent extraction, ethanol at
5560C was found to be the most suitable choice. Fur-
thermore, they concluded that SFE-CO2 was the most
effective method for extracting both -tocopherol and
-oryzanol from rice bran.55
In another study, Lai etal. found that both methanol
and ethyl acetate extracts of japonica rice bran exhibited a
higher -oryzanol content (1.61.8g/kg bran) compared
to the n-hexane extract.56 Using three different solvents,
Chen and Chiu tried to characterize the major phyto-
chemicals and antioxidant properties of taro-scented rice
bran and found that ethyl acetate can extract -oryzanol
more efficiently (1.550.20g/kg rice bran) while the
use of methanol leads to higher yields (15.421.41g/
kg bran).57 Moreover, Rodrigues and Oliveira demon-
strated that ethanol is an efficient solvent for extraction of
-oryzanol and the influence of temperature is important
416 32. -ORYZANOL: ANALYSIS AND CHARACTERIZATION
when a low level of water is added to ethanol.58 In a sec-
ond work on the same subject, Oliveira etal. showed that
it is possible to obtain 15274164mg of -oryzanol/kg of
fresh rice bran when ethanol is used as an extraction sol-
vent and when particular attention is given to solvent
hydration, temperature, solvent-to-rice bran mass ratio,
and stirrer speed.59
Chemical and physical refining of RBO affects the
concentration of -oryzanol in the final product differ-
ently. A complete work on this influence has been carried
out by Gopala Krishna and coworkers.11 Specifically, the
effects of chemical and physical refining as well as the
different processing steps of chemical refining on the
content of -oryzanol were investigated. Initially, the
authors compared physical and chemical refining meth-
ods regarding the concentration of -oryzanol in refined
oil, and found that the oil derived by the physical refin-
ing process retained the original amount of -oryzanol
(1.60% and 1.74%), while the chemically refined oil
showed a considerably lower amount (0.19%). Subse-
quently, they studied the effect of the chemical refin-
ing steps and found that the alkali treatment removed
93.094.6% of the total amount from crude oil, while
the degumming and dewaxing steps removed only
1.1% and 5.9% of the total amount, respectively. On the
other hand, bleaching and deodorization of RBO did not
affect the content of -oryzanol. Finally, the quantitative
analysis of by-products of the chemical refining pro-
cess showed that the soapstock and acid oil contained
high levels of -oryzanol (6.36.9% and 3.37.4% respec-
tively), indicating that they are a good source of these
compounds.11 In another study, Pestana-Bauer etal.
evaluated the contents of -oryzanol and tocols in all the
residues produced during RBO refining. Results showed
that the precipitated soap had the highest -oryzanol
concentration (14.2mg/g, representing 95.3% of the total
-oryzanol in crude RBO) while distillation of the residue
after fatty acid recovery from soap was the best source
of -oryzanol (43.1mg/g, representing 11.5% of the total
-oryzanol in crude RBO).60 Similarly, Scavariello and
Barrera-Arellano explored the influence of temperature
and the molar ratio of sulfuric acid/soaps on -oryzanol
content during the acidulation process of soapstock.
They found that temperature does not significantly
influence the concentration of -oryzanol in the acid oil,
whereas the different ratios of sulfuric acid/soaps gave
concentrations of -oryzanol varying between 3.13% and
3.74%.61
Optimization of the refining process in order to pro-
duce food-grade RBO rich in phytochemicals has been
the subject of several studies as well. Dunford and King
used SFE-CO2 fractionation as an alternative process to
reduce the free fatty acid (FFA) content and minimize
phytosterol loss in RBO. They stated that RBO fractions
with <1% FFA and 1.8% -oryzanol could be obtained
A. OVERVIEW OF RICE AND HEALTH
when particular attention was given to pressure and
temperature conditions.62 Van Hoed etal. reported an
optimized physical refining method to produce RBO
with an acceptable color and high -oryzanol content.
The developed process consisted of an acid degumming,
prebleaching, dewaxing, physical removal of free fatty
acids using packed column technology, a modified wash-
ing step, conventional bleaching, and deodorization.
The refined RBO produced had a color similar to that
obtained from chemical refining, and -oryzanol recov-
ery of 39%.63 Along these lines, Sereewatthanawut and
coworkers reported two-step nanofiltration processing of
crude RBO to produce refined oil enriched in -oryzanol.
The final product was acceptable for consumption (FFA
<0.20wt%) while the -oryzanol level was increased
from 0.95% (in commercial oil) to 4.1wt%.64
Due to its commercial importance, many attempts
have been made to isolate and purify -oryzanol, mainly
from rice industry by-products. There is particular inter-
est in its recovery from RBO soapstock, as is clearly
demonstrated by the approximately 40 patents that have
been filed worldwide for this purpose. Published reports
cited in the literature use a wide range of methods and
techniques, including simple extraction, crystalliza-
tion, and silica-based chromatography, as well as more
advanced procedures such as countercurrent chroma-
tography (CCC). An overview of the different methods
used for the isolation and purification of -oryzanol, as
well as the advantages and limitations of each process, is
presented below.65
Solvent Extraction Processes
Tsuchiya and colleagues reported a process based on
liquidliquid extraction for the isolation of 60% pure
-oryzanol from a high starting amount of RBO (100kg).
This process requires five individual steps, including
two saponification reactions, hydrolysis with HCl, and
two liquidliquid extractions with ether and aqueous
alkali. The low yield of -oryzanol and the number of
steps are the main disadvantages of this process.66 Like-
wise, Masao and Yoshizane reported a patent in which
a two-step leaching process for isolation of -oryzanol
from RBO is used. Initially, the soapstock is leached with
methanol or ethanol and carbon dioxide is transmitted.
As a result, removal of the major impurities and the
soap is accomplished, leaving -oryzanol in the puri-
fied medium. A second leaching step is then required,
and methanol or ethanol washing are used to remove
weak alkali salts from the dry residue. This process led
to efficient removal of impurities from the soapstock
and recovery of 85% pure -oryzanol.67 Takeshi reported
a patented process for obtaining -oryzanol from soap-
stock which includes two main parts and a total of eight
successive steps. In the first part, FFAs were esterified to
 EXTRACTION AND ISOLATION OF -ORYZANOL
methyl esters (FAMEs) and continuously removed from
the soapstock by distillation, while in the second part of
the procedure the residue that remained after distillation
of FAMEs was leached consecutively with n-hexane,
methanol, and methanolic alkali. The drawback of this
process is the high number of steps involved.68 Finally,
Indira etal. have reported a simple, rapid and easy to
scale process for the recovery of -oryzanol from the
saponified and dehydrated soapstock. In this process
-oryzanol was directly extracted from the raw mate-
rial by leaching, using solvents such as ethyl acetate,
acetone, or mixtures thereof. It was noted that the oper-
ating conditions, such as temperature and pH, should
be controlled in order to minimize the degradation of
-oryzanol during processing. The main disadvantages
are the varying purity (3343% w/w) and the low recov-
ery rate (5780% w/w) of the obtained -oryzanol.69
Crystallization Methods
Koji and Tokuo reported a two-step procedure to
purify -oryzanol. Initially, a mixture of alcohols (metha-
nol, propanol, or butanol) and hydrocarbons (n-hexane,
cyclohexane), or toluene was used in a multistage crys-
tallization procedure, while in a second step the obtained
material was submitted to a recrystallization step using
the same solvent mixture. However, the original raw
material, the final purity, and the yield of -oryzanol were
not recorded.70 Similarly, Mingzhi and Yanyan reported
a patent in which a four-step crystallization procedure
for extraction of high purity -oryzanol (98%) from RBO
is described. The second alkali-refining soapstock was
used as a starting material, and a multiphase fractional
crystallization procedure was carried out.71 Along the
same lines, Narayan etal. described a crystallization
process for the recovery of 65% pure -oryzanol from
a -oryzanol enriched fraction. The initial material was
obtained by leaching of pretreated and dehydrated soap-
stock, and treated with mixtures of acetone and metha-
nol in different proportions. With cooling of the eluent
at room temperature mucilaginous impurities were pre-
cipitated, while further cooling to 510C overnight led
to -oryzanol crystallization.72 Another example is the
work of Zullaikah etal.; these authors achieved isola-
tion of -oryzanol from RBO by a two-step crystalliza-
tion process. The first crystallization step resulted in the
removal of mainly triacylglycerol (TG) and steryl esters,
while -oryzanol remained in the liquid phase along with
FFA, monoacylglycerol, squalene, tocols, and phytoster-
ols. At the second crystallization step, the liquid phase
(-oryzanol-rich product) was kept at room temperature
(20.51.5C) for 24h, and after addition of n-hexane as
an antisolvent was further kept at 51C for another
48h and crystal -oryzanol was precipitated. This crys-
tallization process resulted to 9395% pure -oryzanol,
A. OVERVIEW OF RICE AND HEALTH
417
but with a total recovery rate of only 59%.73 Likewise,
starting with RBO soapstock, Kaewboonnum and cowork-
ers have recently reported a method for the recovery of
-oryzanol using a two-step crystallization procedure.
The initial material was obtained from soapstock after
saponification, dehydration, and extraction of residue by
ethyl acetate. At the first crystallization step, a mixture of
ethyl acetate/methanol was used at 30C for 1h, while
the second crystallization step was carried out at 5C for
24h. At the end of the procedure the yield and purity of
-oryzanol were 55.170.59 wt% and 74.604.12 wt%,
respectively. The drawbacks of this process were the low
purity and recovery of the target compounds.5
Silica-Based Chromatographic Methods
Several methods have been also reported for the iso-
lation of -oryzanol using silica-based chromatographic
methods. For instance, Tsuchiya and Okubo reported an
attempt to extract -oryzanol from rice bran acid oil. The
process included, first esterification of FFAs to methyl
esters followed by distillation of the esters, and, sec-
ondly, treatment of the residue by column chromatogra-
phy using a mixture of alcohol and ether as eluent. The
main drawback of this process was the low yield (1.75%),
while the purity of the -oryzanol was not mentioned.74
Likewise, Xu and Godber have used preparative normal-
phase HPLC to isolate -oryzanol of high purity from
crude RBO and, subsequently, reverse-phase HPLC to
separate individual components.75 Lai etal. reported an
efficient chromatographic method to obtain 9098% pure
-oryzanol from RBO using a preparative normal-phase
column chromatography. As the stationary phase a silica
gel packed with small particles (12m) was used, while
the mobile phase (n-hexane and ethyl acetate) was used
in a three-step gradient elution program starting from
85:15 v/v, continuing with 50:50 v/v, and completed
with pure ethyl acetate. As stated by the authors, it is an
easy to scale-up method in order to achieve high recov-
ery (about 90%) of -oryzanol. However, the major disad-
vantage of this procedure was the solubility limit of RBO
(about 3035wt/vol%), which allowed a maximum pro-
duction of 10mg per injection.76 Moreover, Stggl etal.
reported a comparative study for the utilization of C18
and C39 silica stationary phases in the detection and sep-
aration of tocopherols, carotenoids, and -oryzanol in a
single run. According to the results, it was demonstrated
that higher resolution between all target compounds was
obtained using the C30 stationary phase.77
Countercurrent Chromatography (CCC)
Recently, Angelis and coworkers described an advanced
method for the isolation of high purity -oryzanol from
crude RBO using CCC. This process includes one-step
418 32. -ORYZANOL: ANALYSIS AND CHARACTERIZATION
fractionation and isolation of high purity (97%) -oryzanol
from RBO using a non-aqueous biphasic solvent system
(heptaneacetonitrilebutanol 1.8:1.4:0.7, v/v/v). Addi- was achieved. Finally, this fraction was subjected to a
tionally, a fraction containing hydroxylated triterpene
alcohol ferulates or polar -oryzanol was obtained. Scale-
up from the analytical to preparative chromatographic
column was also successfully achieved, revealing the
advantage of CCC for large-scale operations compared to
other isolation techniques.17 To our knowledge, this is the
only study so far concerning the use of CCC for the isola-
tion of -oryzanol.
Combined Approaches
Apart from the abovementioned methods, there
are several studies that incorporate different chro-
matographic methods for the isolation of highly pure
-oryzanol. For instance, Yasuo etal. described a four-
step procedure to recover -oryzanol from RBO. First,
the FFAs were esterified and FAMEs were removed from
RBO by molecular distillation. The remaining unsaponi-
fied fraction was subjected to liquidliquid extraction
using a mixture of n-hexane and tetrahydrofurane. The
tetrahydrofurane phase, in which the unsaponified frac-
tion was partitioned, was concentrated and an amount
of water was added. The -oryzanol was precipitated
and recovered in the aqueous phase. Finally, crystalliza-
tion with n-hexane resulted in highly pure -oryzanol
(98.3%). However this process has the disadvantage
of low recovery of -oryzanol.78 Similarly, Masao and
Yoshizane used RBO as the raw material for the extrac-
tion of -oryzanol via a procedure containing two leach-
ing steps and two liquidliquid extraction steps. For the
leaching, solvents such as trichloroethylene, benzene,
n-hexane, or a mixture of benzene and n-hexane and gas
CO2 were used, resulting in the elimination of impurities
(mainly soap) from the soapstock. Regarding the liquid
liquid extraction phase, the residual mixture obtained
after a second leaching step took place using the same
solvent mixture. This process led to the recovery of
-oryzanol with purity of up to 90% (w/w). The use of
chlorinated or aromatic solvents could be considered the
major negative aspect of this method.67
Seetharamiah and Prabhakar reported a process that
includes liquidliquid extraction, column chromatog-
raphy, and crystallization techniques for the isolation
of -oryzanol from RBO. In the leaching step a mixture
of diethyl ether and methanol was used, and the ether-
rich phase was extracted repeatedly with aqueous alkali.
The alkaline -oryzanol extracts were neutralized with
acetic acid, and -oryzanol was extracted with diethyl
ether. Subsequently, the -oryzanol rich fraction was
subjected to column chromatography on alumina as sta-
tionary phase, and n-hexane, petroleum ether/methanol
(9:1 vol/vol) and diethyl ether/methanol (20:1 vol/vol)
A. OVERVIEW OF RICE AND HEALTH
were used for elution. As a result, a recovery rate of
75.7% (w/w) -oryzanol with a purity of 51.4% (w/w)
two-step crystallization process in order to improve the
purity of -oryzanol. The weakness of this method is the
number of repeated liquidliquid extraction steps and
the column chromatography, which hinders its use for
large-scale purposes.79
Saska and Rossiter described a two-step procedure
for the isolation of -oryzanol from degummed and
dewaxed RBO. Primarily, the use of a simulated moving
bed chromatography separator resulted in enhancement
of -oryzanol, from 1.21.6% to 1215%. Subsequently,
crystallization of the crude product with heptanes led
to 9095% pure -oryzanol, with a recovery rate of
8590%. The drawbacks of this method are the high
investment cost and complexity related to the use of
simulated moving bed chromatography separators.80
Similarly, Das etal described a two-step method for the
isolation of -oryzanol from RBO. At the first step, the
initial material was subjected to vacuum distillation for
FFA elimination. Afterwards, aqueous alkaline hydro-
lysis of the residue was carried out and anionic micel-
lar aggregates containing solubilized -oryzanol were
formed. The addition of calcium ions to this aqueous
micellar aggregate induced instant coprecipitation of
the calcium salts of the fatty acids and the aggregate-
associated -oryzanol. The dried precipitate was then
extracted with ethyl acetate and evaporated. At a second
step, -oryzanol rich residue was purified by silica gel
column chromatography.81
An interesting approach was presented by Rao etal.,
who compared four techniques in a six-step procedure
in order to isolate -oryzanol from RBO. The process
included saponification of the neutral oil present in
the soapstock, leaching of residue, crystallization, and
column chromatography and recrystallization of the
-oryzanol fraction. The overall outcome was the attain-
ment of 90% (w/w) pure -oryzanol, with a recovery rate
of 5670% (w/w). The limitation of this process is the
number of steps required, as well as the use of column
chromatography, which makes the scaling-up very com-
plex.82 Moreover, Kasim etal. described the isolation of
-oryzanol from residue obtained during the production
of biodiesel from RBO using a series of steps in a three-
phase procedure. Initially, the residue obtained during
the production of biodiesel from RBO was subjected to
a degumming and dewaxing procedure, acid-catalyzed
esterification, and vacuum distillation. The derived
residue was then extracted, leading to an increase in
-oryzanol content from 16% to 47%, with a recovery
rate of 97%. Finally, silica gel column chromatography
was used for the purification of -oryzanol to 83.79%,
with a recovery rate of 81.75%, while the overall recov-
ery was estimated to be approximately 69.82%.83
 -ORYZANOL ANALYSIS AND CHARACTERIZATION IDENTIFICATION OF DIFFERENT COMPONENTS
An innovative and easy method for the rapid separa-
tion of -oryzanol in non-aqueous systems was recently
proposed by Kaewchada etal. The process includes
synthesis of molecularly imprinted polymers (MIPs)
with selectivity for -oryzanol. Polymeric materials
were synthesized via a thermal polymerization method,
using -oryzanol as a template, anacardic acid (AnAc)
as a functional monomer, toluene as a porogen, ben-
zoyl peroxide (BPO) as the initiator, and divinylbenzene
as the cross-linker. Different parameters that affect the
absorption capacity of MIPs have been described. How-
ever, further investigations are required in order for this
method to find wider application in the separation of
-oryzanol.84
-ORYZANOL ANALYSIS
AND CHARACTERIZATION
IDENTIFICATION OF DIFFERENT
COMPONENTS
The particular chemical nature of -oryzanol, consist-
ing of numerous components together with their high
quantitative alternations and close structures, compli-
cates considerably its characterization and identification.
Moreover, the lipophilicity of -oryzanol, which could
be considered as a relative polar entity in a highly lipo-
philic environment such as RBO, allows the utilization
of several techniques but at the same time compli-
cates the selection of suitable analytical methods. The
presence of several substrates, such as crude oil and
RBO fractions, etc., as well as the complicated isola-
tion procedure required for the delivery of high purity
-oryzanol further hinders its analysis; this is of major
importance due to the increasing interest in -oryzanol
and its multiple applications. Therefore, since its initial
identification6 several analytical methods have been
proposed for the determination of different -oryzanol
constituents and for their quantitation in different
materials. It is also noticeable that new components are
constantly being suggested.13,17,85,86 The first published
reports regarding -oryzanol were focused mainly on
the separation and characterization of -oryzanol com-
ponents, incorporating several chromatographic meth-
ods such as thin layer chromatography (TLC), liquid
chromatography (LC), gas chromatography (GC), GC-
mass spectrometry (MS), and high performance liq-
uid chromatography (HPLC) mainly reversed phase
(RP).8,9,87 Regarding identification, much information
was first derived from research studies carried out on
steryl and triterpene alcohol ferulates isolated from
other sources. CG and GC-MS were used as methods
of choice for the identification thereof, usually after
derivatization.88,89 Later on, most studies were related
to the detection of -oryzanol in multiple rice-related
A. OVERVIEW OF RICE AND HEALTH
419
materials and in particular in RBO, and its separation
from other components such as lipids, tocopherols,
and tocotrienols, while a number of methods were sug-
gested for -oryzanol quantitation.
One of the first complete methods for the separation,
identification, and quantitation of -oryzanol was pro-
posed by Rogers etal. in 1993. Specifically, a reversed
phase (RP) high-performance liquid chromatography
(HPLC) device connected to a photodiode array detec-
tor (PDA) was utilized. The mobile phase consisted of
acetonitrile/methanol/isopropanol/water (45:45:5:5
by volume) and was linearly modified to acetonitrile/
methanol/isopropanol/water (50:45:5 by volume) for 4
minutes, after the first 6 minutes, and was kept constant
for 15 minutes before a return to the initial conditions.
The selected wavelength for monitoring -oryzanol was
325nm. Moreover, chemical ionization (CI) MS was incor-
porated for the elucidation of the structure of -oryzanol
components. Cycloartenyl ferulate, 24-methylene cyclo-
artanyl ferulate, campesteryl ferulate, -sitosteryl feru-
late, and cycloartanyl ferulate were first proposed as
the major constituents.90 Additionally, the developed
methods were applied to several fully processed edible
RBOs from different manufacturers. After approximately
a year, and due to the lipophilic nature of RBO, a new
method was suggested for the same purposes using nor-
mal phase (NP)-HPLC.91 Five commercial silica-based
columns were assayed and several chromatographic
parameters, such as mobile phase, temperature, and
flow rate, were optimized. For the same reason, a method
using HPLC connected to evaporative light-scattering
detector (ELSD) has been proposed.92 In parallel, some
reports regarding the qualitative and quantitative char-
acterization of -oryzanol in biological fluids appeared
more than three decades ago, following the first evidence
regarding its biological properties, and were related to
its metabolism and absorption in animals93,94 as well as
the determination of ferulic acid, a main metabolite of
-oryzanol.95,96 Of high importance is the work of Nor-
ton, who investigated the separation, isolation, and
identification of steryl and triterpene alcohol ferulates,
cinnamates, and coumarates in corn bran and rice bran,
published in 1994 and 1995. An optimized RP-HPLC-
PDA method was proposed for their separation and
quantification, and a GC-MS method was developed,
after derivatization, for the identification of -oryzanol
components.97,98 A very significant contribution was that
of Xu and Godber, mainly to the separation of high purity
-oryzanol from RBO and the identification of additional
minor constituents. Using a combination of NP and
RP-HPLC, the isolation of several compounds of satisfac-
tory purity was achieved, while GC-MS using the electron
impact (EI) ionization method was utilized for the iden-
tification thereof. More than 10 different steryl ferulates
were suggested: 7-stigmastenyl ferulate, stigmasteryl
420 32. -ORYZANOL: ANALYSIS AND CHARACTERIZATION
ferulate, cycloartenyl ferulate, 24-methylenecycloartanyl
ferulate, 7-campestenyl ferulate, campesteryl ferulate,
7-sitostenyl ferulate, sitosteryl ferulate, compestanyl fer-
ulate, and sitostanyl ferulate. Three (cycloartenyl ferulate,
24-methylenecycloartanyl ferulate, and campesteryl feru-
late) were found to be the major components of -oryzanol,
verifying previous reports. Moreover, useful structural
information was derived for the identification of steryl fer-
ulates after TMS (trimethylsilylane) derivatization based
on their fragmentation patter, via GC-EI-MS analysis.75 It
is noteworthy that the work of Xu and Godber was inte-
grated to a significant degree by Akihisa etal. in 2000.22
Six novel steryl ferulates, two trans- and four cis-ferulates,
together with five known trans-ferulates and one known
cis-ferulate, respectively, were isolated using preparative
TLC, NP-HPLC, and RP-HPLC. The preparation of syn-
thetic derivatives and the corresponding free forms have
assisted considerably in the identification procedure and
the evaluation of their anti-inflammation properties, and
resulted in valuable NMR data. It is important to note that
this work unambiguously confirmed the presence of cis-
derivatives in RBO, which could comprise artifacts of the
refining procedure.99,100
Generally, using 1H NMR analysis, the identification
of steryl ferulates among other chemical classes can be
performed easily if the purity level is satisfactory. Based
on the structural characteristics of the core skeleton, the
signals could be divided in two groups: one correspond-
ing to the aromatic part of the molecule (ferulate), and
the other corresponding to the triterpenoid part (sterol
and/or tritepene alcohol).101 The protons of the two
parts of the molecule are resonated in different chemi-
cal shift areas. In particular, the aromatic ones are reso-
nated in low fields (approximately 6.57.5ppm) and the
aliphatic ones in high fields (0.52.5ppm). The signals
of the double bonds of the aliphatic part, which are
resonated in the area between (4.55.5ppm), are very
characteristic and could be utilized as indicative in the
identification process. Also, rather characteristic are the
signals of the double bond of the ferulate moiety.102 The
signals of these two protons of the double bond are res-
onated in low fields, separately, as two double peaks.
In particular, the proton closer to the aromatic ring
appears very deshielded (7.6ppm) while the other one
is resonated in higher fields (@6.3ppm), in respect to
the deuterated solvent used. However, most indicative
is the coupling constant value (J) of these peaks, which
could be used for the identification of trans- and cis-
ferulates since they are at approximately 1516Hz and
1213Hz, respectively. Finally, quite indicative are the
signals of the methyl groups, which could be used for
the determination of subgroups and further structural
details.22,89,103
Furthermore 13C NMR data could be rather useful for
the structure elucidation of steryl ferulates. Based on the
A. OVERVIEW OF RICE AND HEALTH
degree of substitution (mainly methyl groups), the num-
ber of rings (4- or 5-rings derivatives) and the length of
the side chain (usually C5 or C6), the total number of car-
bon signals detected are 40, in average (37 to 41 for the
identified RBO steryl ferulates so far). Similarly to the
1H NMR spectra, the signals could be split in two main
groups. The first set of signals (approx. 30) is resonated
in high fields (10 to 60 ppm, approx.) corresponding to
the triterpenoid part of the basic structure while the rest,
approximately 10 signals are appeared rather deshielted
(100 to 170 ppm, approx.) and correspond to the ferulic
moiety. Specifically, characteristic are the signals of the
carbonyl group ( 165170 ppm), the oxygenated meth-
ane carbon ( 80 ppm) as well as the methyl group of
the ferulate resonated at around 55 ppm. Nevertheless,
for detailed structure information, 2D NMR analysis is
required.102104
The available structural information was enriched by
the contribution of Fang etal., who gave new insight in
MS-based identification of steryl ferulates. The use of an
Ion Trap (IT) analyzer equipped with an electrospray
ionization probe (ESI), hyphenated to a HPLC enabled
the determination of several MS/MS fragments and
revealed fragmentation partners of steryl ferulates.13
Both ionization modes, negative and positive, were
used with the last being more effective. Most of the data
regarding useful fragments of steryl ferulates were in
accordance with previous studies using conventional
GC-EI-MS platforms. However, the major advantage of
this approach is that no treatment of the sample e.g. RBO
(saponification) and/or derivatization (sililation, acety-
lation) is required prior analysis. Furthermore, hydrox-
ylated derivatives of steryl ferulates were suggested
for the first time and were further analyzed afterwards
from the same group.13,105 The same year, an interesting
approach was proposed from Miller etal. for the rapid
analysis and comparative studies of -oryzanol, in dif-
ferent rice varieties using coupled liquid-gas chromatog-
raphy (LC-GC).106 Pre-separated fractions using HPLC,
of -oryzanol were transferred on line to GC analysis
while the identity of the detected compounds was also
confirmed by off-line GC-MS.
The following years, several analytical methodologies
focusing mostly to the simultaneous characterization
of different chemical classes of secondary metabolites
occurring in RBO were suggested. Different analytical
parameters, alternative platforms and/or materials were
incorporated for this reason.28,107,108 For instance, Stoggl
etal. reported an approach for the concurrent analysis
of -carotene, tocopherols, and -oryzanol, using RP-
HPLC-PDA, in a single run of 20 min, using methanol/
tert-butyl methyl ether (75:25 v/v) as a mobile phase. A
study comparing HPLC C18 and C30 columns, was also
carried out and the last found more efficacious. Addi-
tionally, an IT analyzer equipped with an atmospheric
 -ORYZANOL ANALYSIS AND CHARACTERIZATION IDENTIFICATION OF DIFFERENT COMPONENTS
pressure chemical ionization (APCI) probe was used
for the verification of steryl ferulates identity while
both direct infusion and LC-MS methodologies were
employed.77 Another example is the RP-HPLC-PDA
method proposed by Huang and Ng, in 2011 aiming to
the simultaneous determination of tocopherols, tocotri-
enols and -oryzanol in rice; however, focusing mainly
on the first two classes.109 The last 3 years limited addi-
tional methods have been proposed giving new alterna-
tives at -oryzanol analysis and characterization. Zaima
etal., in 2010 presented a matrix-assisted laser desorp-
tion/ionization (MALDI)-imaging mass spectrometry
(IMS) method for analyzing rice grains and the distri-
bution of different metabolites. According to this study,
-oryzanol is localized in the bran (germ and seed coat)
together with phoshatidylcholine and phytic acid while
-tocopherol is mainly present in the germ, especially in
the scutellum.110
Furthermore, in 2011 a new LC-MS method was sug-
gested by Angelis etal., employing a high resolution
(HR) MS analyzer and, specifically, the relatively newly
invented Orbitrap analyzer (Orbital trap), equipped
with an APCI probe, in both modes. To our knowledge,
this was the first time that a HRMS platform was uti-
lized for the analysis of steryl ferulates, verifying previ-
ous findings but also revealing new structural data for
the identification of -oryzanol. Additionally, a new class
of -oryzanol, the so-called polar -oryzanol, was deter-
mined, eluting similarly to -oryzanol as a set of com-
pounds and presenting higher hydrophilicity. According
to the authors, the utilization of APCI methods in both
modes assisted the detection of possible impurities and,
specifically, the presence of glycerol esters, which are
highly abundant in RBO and usually co-eluted with
-oryzanol components.17
In general, considering the elucidation of the struc-
ture of steryl ferulates using MS, most of the information
available has been derived by GC-MS methodologies
using traditional derivatization techniques such as acet-
ylation or silylation of -oryzanol or its basic compo-
nents after isolation. Interpretation of the spectra usually
follows primarily the identification of diagnostic peaks
of the ferulic moiety (with or without the derivatization
unit, e.g., Ac, TMS) and afterwards identification of the
triterpenoid part based on the corresponding fragmenta-
tion pattern.88 In particular, under EI conditions, char-
acteristic ions at m/z 177 and 193 (underivatized forms)
are observed as indicative for the ferulic moiety, which
could also be used for the differentiation of steryl and
stanyl ferulates. Also, in most cases the fragment ions
which correspond to the triterpenoid part of the mol-
ecule are present after cleavage of the ferulic acid. Simi-
lar observations have been made by Das etal., using the
LSIMS method, in positive mode, while they also noticed
the presence of pseudomolecular ions corresponding
A. OVERVIEW OF RICE AND HEALTH
421
to adducts with sodium.81 Moreover, the detection of
[M-C10H10O4+H]+ ions and their utilization as being
indicative for the identification of steryl ferulates was
also confirmed from Stoggle etal., using LC-APCI-MS
methods without prior derivatization.77
On the other hand, when using atmospheric pressure
ionization (API) methods such as ESI or APCI, the frag-
mentation is less extensive compared to hard ionization
techniques such as EI or chemical ionization (CI), and
specifically in the negative mode pure spectra are deliv-
ered most of the time. More detailed information could
be derived by the use of LC-MS-MS methods in both
modes. In the negative mode, the ions are characteristic;
these are formed after the elimination of one and/or two
methyl groups from the ferulic and the triterpenoid part,
respectively, while the [feruloyl] fragment ion is also
evident. In contrast, no fragment ions corresponding
to the triterpenoid part of the molecules were detected,
implying that the negative mode is less pronounced
for the identification of steryl ferulates. In the positive
mode, basic ions are the intact alcohol moiety (triterpene
alcohols or sterols), which are derived from the cleavage
of feruloyl and alcohol moieties. Also indicative are the
ions yielded by the loss of different numbers of methyl
and methylene groups.
These data, as well as other MS information regard-
ing more in-depth characterizations of steryl ferulates,
have been discussed by Fang etal.13 In particular, the
author reported that cis-isomers have longer retention
times than the corresponding trans-isomers using C18
RP-HPLC (e.g., campesterol cis-ferulate vs campesterol
trans-ferulate). Also highlighted was the presence of
hydroxylated steryl ferulates based on the highly intense
[M+H-194-H2O]+ ions. Moreover, they reported for the
first time the presence of caffeate esters of STAFs in RBO
based on the presence of fragment ions at m/z 179 in neg-
ative CID spectra and [M+H-180]+ ions for intact alcohol
moieties (triterpene alcohols or sterols) in positive mass
spectra together with the absence of [MHMe] ions.
Along the same lines, the utilization of HR-MS plat-
forms for the identification of steryl ferulates could be
considered of high importance. Accurate mass mea-
surements, high resolution spectra, and additional
spectrometric features provide high confidence in the
identification procedure.111 Until now, there is only one
publication, by Angelis etal., that reports the character-
ization of steryl ferulates in RBO using the LC-HR-MS
platform, equipped with an APCI probe, in both modes.
Utilization of the extraction ion method (XIC chro-
matograms) has revealed the presence of both cis- and
trans-isomers and therefore facilitated significantly the
characterization of -oryzanol. Moreover, the use of a
suggested elemental composition (EC) for every ion of
interest measured accurately (m23 ppm) together
with ring double bond equivalent (RDBeq) values has
422 32. -ORYZANOL: ANALYSIS AND CHARACTERIZATION
led to identification of steryl ferulates with high confi-
dence.17 Furthermore, additional possible derivatives
were suggested by the authors, while the ion [M+H
H2O]+, present in full scan spectra, was proposed as
indicative for the identification of polar -oryzanol.
Apart from the traditional and most commonly utilized
analytical methods, a few others have been incorporated
for the analysis, quantification, and characterization of
-oryzanol for various purposes. For instance, Deepam
and coworkers reported the development of a high-per-
formance thin layer chromatography (HPTLC) method
for the constituents of RBO112, while Kumar etal. pro-
posed a thin-layer chromatographic (TLC) method for
the detection of RBO in other vegetable oils monitor-
ing -oryzanol components.113 Furthermore, alternative,
non-chromatography-based techniques have also been
proposed for the determination of -oryzanol in RBO.
For instance, spectrophotometric methods such as fixed
wavelength, and second-derivative and multicompo-
nent analysis, have been reported for the quantitation of
-oryzanol.114
After reviewing the previous and current trends in
the analysis and characterization of -oryzanol, it will
be useful to cite and briefly discuss the most common
applications where these methods are utilized. Thus,
the methodologies mentioned have been used in the
evaluation of the extraction and isolation procedure of
-oryzanol or its components from different materials,
such as RBO and soapstock, since the efficient and fast
procumbent thereof remains an important challenge,
mainly for industry.5,56,59,101,115117 Known or slightly
modified methods have also been incorporated for qual-
ity assessment of the RBO production procedure and
the corresponding derived materials, as well as for the
appraisal of oils from different sources and for adul-
teration issues.118,119 In particular, different parameters,
mainly regarding the refining process, related to the con-
tent of -oryzanol still comprise an important research
topic.11,60,63,120-126 Numerous studies dealing with the
stability of -oryzanol, mainly during storage or cook-
ing,127129 have been performed, for the identification
of optimal or alternative sources130 and the isolation of
-oryzanol, as well as comparative studies between dif-
ferent rice varieties,39,40,42,43,131136 commercial rice vari-
eties,41,137 and conventional and organic rice102 mutant
species.138140 An interesting research topic would
include localization of -oryzanol in the different parts
of rough rice seed,47,141 its content in paddy and brown
rice,50,142 and its quantitative differentiation during
maturation and development.44,46,49,52,143145 Finally, the
existing methods have also been incorporated to detect
and or quantify -oryzanol in biological samples in order
to further investigate its biological properties and role;
for instance, the work by Lubinus etal. is related to the
fate of steryl ferulates upon consumption by healthy
A. OVERVIEW OF RICE AND HEALTH
humans.146 However, such studies are rather limited and
further investigation is required.
PHARMACOLOGICAL ACTIVITIES AND
APPLICATIONS OF -ORYZANOL
Since the discovery of -oryzanol in the 1950s, numer-
ous studies have been performed exploring the pharma-
cological potentials of -oryzanol.4,26,147 Widely used in
Asia as a therapeutical agent to treat clinical disorders
involved with the menopause,148 stimulation of the seba-
ceous glands,149 or ulcers,150 -oryzanol has attracted
much interest throughout the world. According to Cicero
and Gaddi,151 early studies on the biological profile of
-oryzanol started with the work by Nakamura152 in the
1960s regarding the influence of -oryzanol on hepatic
cholesterol biosynthesis. Since then, according to the
PubMed database, more than 180 research articles have
been published, presenting a wide spectrum of biologi-
cal activities. In addition, several reports investigating
the biological properties of -oryzanol rich extracts and
commercial or purified -oryzanol, using various invitro
or invivo models, have also been presented.
The larger proportion of these biological studies con-
cerns the antioxidant activity of this -oryzanol, due
to the presence of the phenol moiety in its composi-
tion. By definition, reactive oxygen species (ROS) are
free radicals which, when they are overproduced in
the organism, may cause oxidative stress by oxidation
of biological macromolecules such as proteins, nucleic
acids, and cell membranes. This oxidative damage is the
origin of many chronic diseases, and cell aging. Thus,
over recent decades the research into new antioxidants
has been considerably increased.
In this context, due to the presence of the ferulic/
caffeic acid part, which enables donation of electrons
and destroys the action of free radicals, -oryzanol has
been widely studied for its antioxidant properties.153155
For example, Kims work shows that -oryzanol inhib-
its pyrogallol autoxidation and is more effective than
the well-known synthetic antioxidants (BHA, BHT,
and TBHQ) against hydroperoxide formation.156 More-
over, Hiramitsu etal. have suggested that -oryzanol
inhibits lipid peroxidation induced from porcine reti-
nal homogenates using ferric ion or UV light,157 and
thus could be employed in retinotoxic evaluation of
ophthalmic drugs. On the other hand, Xu and Godber
were more interested in the antioxidant properties of
the individual three major compounds of -oryzanol
(cycloartenyl ferulate, 24-methylenecycloartanyl, and
campesteryl ferulate), using a linoleic acid-based per-
oxidation model. Their results indicated that these com-
ponents are able to significantly reduce the production
of hydroperoxides.158
 PHARMACOLOGICAL ACTIVITIES AND APPLICATIONS OF -ORYZANOL
The antioxidant activities of -oryzanol against cho-
lesterol oxidation have also been investigated.159 Choles-
terol oxidation products (COPs) lead to the formation of
several toxic atherogenic, mutagenic, and carcinogenic
compounds,160 which cause cell membrane damage and
are responsible for many pulmonary and cardiovascu-
lar diseases.161,162 It has been shown that -oryzanol is
capable of reducing the production of COP considerably,
with higher activity than that of vitamin E.163 It is note-
worthy that a significant correlation between oxidative
stress and various diseases, such as malignancies, diabe-
tes, atherosclerosis, chronic inflammation, human immu-
nodeficiency virus (HIV) infection, ischemia reperfusion
injury, and sleep apnea has been highlighted.164 Overall,
given this background, much interest has focused on
investigation of the therapeutic properties of -oryzanol
against several health complications and diseases, using
numerous models and assays.
Anti-Ulcer Effect
In the 1970s1980s, the anti-ulcerogenic properties of
-oryzanol were extensively investigated in Japan, using
rat models. Nevertheless, further studies are needed to
identify its inhibitory effect and establish its real clinical
efficacy.165168
Anti-Inflammatory Effect
Recently, many researchers have investigated the
capacity of -oryzanol to treat inflammation. -Oryzanol
has been found to inhibit the increase in swelling of the
hind paw in adjuvant-induced arthritis in rats in a dose-
dependent manner (1100mg/kg),169 and to possess a
strong anti-inflammatory effect on sodium dextran sul-
fate-induced colitis170 and ethanol-induced liver dam-
age171 in mice, via inhibition of NF-B activity.
Anti-Allergic Effect
Oka and colleagues have demonstrated, for the first
time, a possible anti-allergenic effect of -oryzanol using
a fraction extracted from domestic Japanese rice, accord-
ing to the Bligh and Dyer method. Using the passive
cutaneous anaphylaxis (PCA) reaction model on rats, the
authors observed a significant anti-allergic effect deter-
mined by the inhibition of mast cell degranulation.172
Moreover, using the same methodology they also assayed
the effect of major components of the fraction, such as
cycloartenyl ferulate (28.2%), 24-methylene cycloartanyl
ferulate (22.4%), -sitosteryl ferulate (12.3%), and cyclo-
branyl ferulate (<10%), and similar inhibition of mast
cell degranulation with a greater potential for cyclobra-
nyl ferulate was assisted. Finally, they investigated the
possible mechanism of action proposing that -oryzanol
is able to capture immunoglobulin E (IgE), preventing
its cross-linking to the high-affinity IgE receptor (FcRI),
involved in the allergic disorder.
A. OVERVIEW OF RICE AND HEALTH
423
Antidiabetic Effect
Several experiments have been conducted in order to
analyze the potential of -oryzanol as a therapeutic agent
against diabetes mellitus. In different trials, mainly car-
ried out on diabetic rats, -oryzanol was found to pos-
sess an antidiabetic effect, improving insulin sensitivity
and reducing the blood glucose level.173175
Anticancer Properties
-Oryzanol and its components have been evaluated
for their anticancer properties. Yasukawa etal. reported
an inhibitory effect of the four major components of ory-
zanol (cycloartenyl ferulate, 24-methylene cycloartanyl
ferulate, campesteryl ferulate and sitosteryl ferulate) on
tumor promotion in two-staged carcinogenesis in mouse
skin.176 Furthermore, the recent work by Kim has shown
that -oryzanol significantly reduced the tumor mass in
mice inoculated with CT-26 colon cancer. Oral adminis-
tration of 1% -oryzanol resulted in a dose-dependent
reduction of the tumor growth by 44% without affecting
the weight of other organs.177 In addition, some studies
have been performed in order to explore safety regarding
the carcinogenic properties of -oryzanol, using F344 rat
and B6C3F1 mouse lung carcinogenesis models.178,179 A
tumor progression effect was observed with -oryzanol,
but it was weak and occurred only with high doses.180,181
Antihyperlipidemic Effect
Owing to the structural analogy of -oryzanol com-
pounds with cholesterol, another important research area
concerns its ability to lower cholesterol levels. The first
scientific study on this topic was carried out in humans
in 1970. Specifically, Suzuki and Oshima182 observed a
decrease of total cholesterol (TC), within 7 days, in the
plasma of 50 healthy young women who consumed 60g
of a combination of 70% RBO and 30% safflower oil.
Later, in 1982, Ishihara etal.148,183 focused particularly on
-oryzanol, showing that after four to eight treatments
of 300mg of this component per day in hyperlipopro-
teinemic subjects, there was a decrease in total choles-
terol (TC), low density lipoprotein cholesterol (LDL-C),
and triglyceride (TG) plasma levels, together with an
increase in high density lipoprotein cholesterol (HDL-C)
concentration, with no side effects. Similar results have
been published since in humans,184189 rats,190193 rab-
bits,194 and hamsters.195197 This information is well
reviewed by Cicero and Gaddi,151 with particular atten-
tion to the treatment of hyperlipoproteinemias, indicat-
ing that -oryzanol could be utilized as a therapeutic
agent for hyperlipidemia and atherosclerosis.198,199
Effect on Menopausal Disorders
In Japan, two studies have investigated the effect of
-oryzanol on menopausal disorders. In the first, in the
1960s, 100mg of -oryzanol was administrated to 13
424 32. -ORYZANOL: ANALYSIS AND CHARACTERIZATION
women who had undergone hysterectomy (surgical
menopause) three times a day for 38 days. According
to the findings, 67% of the women reported a signifi-
cant reduction in menopausal symptoms such as hot
flushes.200 In 1982, in another study, 40 women with
climacteric disturbances were administered 300mg of
-oryzanol, daily for 48 weeks. In 90% of the cases a gen-
eral improvement concerning reduction of menopausal
disorders was observed.148 Since then, studies have been
conducted investigating the effect on -oryzanol on
menopausal disorders compared with other approaches
such as acupuncture.201-204 Even if acupuncture presents
a better outcome, Tian indicated that a combined use of
acupuncture and Chinese medicine is more effective for
treating the menopausal syndrome.205
APPLICATIONS
As discussed previously, -oryzanol exhibits a wide
range of biological activities as a natural antioxidant
product. Thereby, it has found numerous applications as
a sunscreen and as an anti-aging agent in the cosmetics
industry, as well as being a natural additive to improve
food stability and a pharmaceutical raw material. For
example, due to its antioxidant properties, -oryzanol is
widely employed and patented as a sunscreen agent in
cosmetic formulations. A non-chemical sunscreen com-
position including -oryzanol together with proantho-
cyanidins, ferulic acid, titanium oxide, and Scutellaria
extract has been patented.206 Recently, -oryzanol has also
been incorporated in an aqueous cosmetic composition
intended for the photoprotection of skin and hair against
UV radiation.207 Furthermore, Manosroi has investi-
gated the anti-aging effect of creams and gels containing
rice bran bioactive compounds including -oryzanol on
human skin.208 Promising results regarding the amelio-
ration of thickness, roughness, and elasticity of the skin
have indicated the potential of -oryzanol for protection
against skin aging. Apart from its application as a skin
protecting agent, -oryzanol has also been introduced as
antioxidant in preparations for eyebrows and eyelashes,
in skin cream, shampoo, and lip balm, and in nail color
products and products for the surrounding skin.12,209
Additionally, due to -oryzanols ability to signifi-
cantly reduce production of toxic cholesterol oxidation
products (COPs) it has been used as a food additive.
During the cooking process or storage of foods, forma-
tion of COPs occurs with the action of air, light, or heat.
In this context, it has been reported that the addition of
-oryzanol delayed the formation of COP in refrigerated
cooked beef210 and improved the oxidative stability of
vegetable oils at frying temperatures.154,211 Additionally,
Khuwijtjarus works have revealed a possible degrada-
tion of -oryzanol in stripped rice bran oil during thermal
A. OVERVIEW OF RICE AND HEALTH
oxidation.212 Accordingly, the effect of -oryzanol micro-
encapsulation, aiming to achieve stability against heat-
induced lipid oxidation, has been studied.213,214 In
exploring the antilipoperoxidation efficacy of -oryzanol
when incorporated into nanosponges, it was found that
-oryzanol retains a significant antilipoperoxidative
activity even when it is encapsulated. Consequently,
encapsulation could be used to protect -oryzanol from
photochemical degradation and thus the loss of its anti-
oxidant effect.10 This high resistance to heat resulted in
the approval and classification of -oryzanol in Japan as
an oxidation inhibitor in the Food Additive List, and
therefore several food stabilization techniques using
-oryzanol have been patented.
Another activity that is attributed to -oryzanol con-
cerns its metabolic effect on the body. Throughout the
world -oryzanol is commonly used by athletes and
bodybuilders as a sports supplement, with many papers
and websites reporting muscle bulk growth in athletes
by the increase in testosterone production and stimu-
lation of human growth hormone release. However, as
pointed out by several authors,215 there is no solid valid
scientific evidence for these effects, and the performance
claims that are advertised are only supported by the
conviction from athletes that -oryzanol is an excellent
ergogen. Faced with this lack of data, two studies have
focused on the possible ergogenic effects of -oryzanol,
but neither has supported this assertion. According to
Wheeler,216 -oryzanol is poorly absorbed and its intra-
venous or subcutaneous injection in rats was found to
reduce hormone synthesis and release, while an increase
in the release of catecholamines, dopamines and norepi-
nephrine in the brain was also observed, leading to the
conclusion that this metabolic milieu may actually reduce
testosterone production. Fry studied the improvement
in muscular power or strength of weight-trained males
who consumed 500mg/day of -oryzanol, after 9 weeks
of an endurance exercise program.217 No significant dif-
ferences between the supplemented and the control pla-
cebo groups were observed for measures of circulating
concentrations of hormones, minerals, binding protein,
or blood lipids, suggesting that more research is needed
regarding this possible anabolic effect.
CONCLUSION
Unquestionably, -oryzanol is a natural entity of high
scientific interest and industrial value. The significant
health-beneficial effects of -oryzanol, as well as its pleo-
tropic nature, have resulted in numerous applications
in nutrition, medicine, and cosmetics. Nowadays, two
main research topics related to -oryzanol concentrate
the interest of the scientists. The first concerns charac-
terization of -oryzanols components, and biological
 References
evaluation of the activity of both -oryzanol and its com-
ponents while the second focuses mainly on the efficient
extraction, isolation, and exploitation of its commercial
potentials. Several new techniques and methodologies
have contributed considerably to both research axes in
the recent years. Sophisticated analytical concepts have
been incorporated for the detection and elucidation of
-oryzanols constituents and their isolation, as well as
its fast, simple, and reproducible production. However,
more effort should be invested in the unambiguous and
complete characterization of -oryzanol, while purity
issues still remain relatively unresolved. Furthermore,
attention should be concentrated on the assessment of
-oryzanols pharmacological properties and explora-
tion of its biological role.
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